Abstract DNA helicases are ATP-dependent molecular motors that unwind duplex DNA to form the single stranded (ss) DNA intermediates required for genome maintenance in all organisms. Defects in DNA helicases are responsible for a number of human diseases. We are studying the mechanisms of DNA unwinding and ssDNA translocation of a multi-subunit DNA helicase/nuclease, E. coli RecBCD, which functions in repair of DNA double strand breaks and recombination. RecBCD is a hetero-trimeric complex containing two superfamily 1 (SF1) helicase/translocase motors (RecB, a 3' to 5' motor and RecD, a 5' to 3' motor) that move with different rates while part of the same complex, but undergo a switch in relative rates after the RecC subunit recognizes an 8 nucleotide ssDNA sequence, called ?chi?. The nuclease activity of RecBCD is also changed dramatically due to an allosteric effect of chi recognition. Despite extensive study, the mechanism of RecBCD-catalyzed DNA unwinding is not understood. There is also little known about how the two motors communicate within RecBCD and the allosteric regulation of its motor and nuclease activities by chi. We have developed novel ensemble fluorescence assays that enable us to monitor ssDNA translocation of the two motors independently. This led to our discovery that, in addition to its primary 3' to 5' translocase, RecBC (without RecD) also possesses a previously unrecognized secondary translocase activity that moves RecBC along the opposite DNA strand. We also recently discovered that RecBCD can unwind duplex DNA processively even in the absence of ssDNA translocation by the canonical RecB and RecD motors indicating that DNA melting and ssDNA translocation are separate processes. We have identified two domains within the enzyme that enable such activity. Our goals are to: 1- understand the mechanism by which RecBC and RecBCD can unwind duplex DNA processively in the absence of ssDNA translocation by its canonical motors, 2- understand the allosteric regulation of DNA unwinding and ssDNA translocation by the RecB nuclease domain and chi, and 3- probe the conformational changes/domain movements within RecBCD and RecBCD-DNA complexes that occur during its activities. Thermodynamic, transient kinetic, structural and single molecule approaches (fluorescence and optical tweezers) will be used to obtain a molecular understanding of the kinetic mechanism(s) by which this complex multi-motor enzyme translocates along and unwinds DNA and is regulated. Such studies will provide new insight into nucleic acid motor enzymes that are essential for the maintenance of all genomes.